Eric Woolf

2011 White Paper on Recent Issues in Bioanalysis and Regulatory Findings from Audits and Inspections

The 5th Workshop on Recent Issues in Bioanalysis (WRIB) was organized by the Calibration and Validation Group as a 2-day full immersion workshop for pharmaceutical companies, CROs and regulatory agencies to discuss, review, share perspectives, provide potential solutions and agree upon a consistent approach to recent issues in the bioanalysis of both small and large molecules. High quality, better compliance to regulations and scientific excellence are the foundation of this workshop. As in the previous editions of this significant event, recommendations were made and a consensus was reached among panelists and attendees, including industry leaders and regulatory experts representing the global bioanalytical community, on many hot topics in bioanalysis. This 2011 White Paper is based on the conclusions from this workshop, and aims to provide a practical reference guide on those topics.

Workshop Report: Crystal City V—Quantitative Bioanalytical Method Validation and Implementation: The 2013 Revised FDA Guidance

In September 2013, the FDA released a draft revision of the Bioanalytical Method Validation (BMV) Guidance, which included a number of changes to the expectations for bioanalysis, most notably the inclusion of biomarker assays and data. To provide a forum for an open, inclusive discussion of the revised draft BMV Guidance, the AAPS and FDA once again collaborated to convene a two-and-a-half day workshop during early December 2013 in Baltimore, MD, USA. The resulting format embodied extensive open discussion and each thematic session included only brief, concise descriptions by Agency and industry representatives prior to opening the floor discussion. The Workshop was built around four thematic sessions (Common Topics, Chromatographic, Ligand-Binding Assays, and Biomarkers) and a final session with international regulators, concluding with a review of the outcomes and recommendations from the thematic sessions. This Workshop report summarizes the outcomes and includes topics of agreement, those where the FDA will consider the Industry’s perspective, and those where the workshop provided a first open dialogue. This article will be available to the bioanalytical community at http://www.aaps.org/BMV13.

Determination of sitagliptinin human plasma using protein precipitation and tandem mass spectrometry

A simple offline LC-MS/MS method for the quantification of sitagliptin in human plasma is described. Samples are prepared using protein precipitation. Filtration of the supernatants through a Hybrid-SPE-PPT plate was found to be necessary to reduce ionization suppression caused by co-elution of phospholipids with sitagliptin. The sitagliptin and its stable isotope labeled internal standard (IS) were chromatographed under hydrophilic interaction chromatography conditions on a Waters Atlantis HILIC Silica column (2.1 mm x 50 mm, 3 microm) using a mobile phase of ACN/H(2)O (80/20, v/v) containing 10 mM NH(4)Ac (pH 4.7). The sample drying after protein precipitation due to high organic content in the sample is not necessary, because HILIC column was used. The analytes were detected with a tandem mass spectrometer employing a turbo ion spray (TIS) interface in positive ionization mode. The multiple reaction monitoring (MRM) transitions were m/z 408-->235 for sitagliptin and m/z 412-->239 for IS. The lower limit of quantitation (LLOQ) for this method is 1 ng/mL when 100 microL of plasma is processed. The linear calibration range is 1-1000 ng/mL for sitagliptin. Intra-day precision and accuracy were assessed based on the analysis of six sets of calibration standards prepared in six lots of human control plasma. Intra-day precision (RSD%, n=6) ranged from 1.2% to 6.1% and the intra-day accuracy ranged from 97.6% to 103% of nominal values.

Building the Global Bioanalysis Consortium – working towards a functional globally acceptable and harmonized guideline on bioanalytical method validation

Although harmonization of bioanalytical method validation (BMV) and application has been disxad cussed at scientific forums on several occasions, it was not until the European Bioana lysis Forum (EBF) conference in December 2009 [1], where the European Medicines Agency’s (EMA) intended draft bioanalytical method validation guidexad line [101] was discussed, that the bioanalytical scientists present expressed their strong wish for international harmonization. CT Viswanathan (US FDA/Center for Drug Evaluation and Research [CDER]) and J Welink (Dutch Medicines Evaluation Board [MEB] for EMA), present at the meeting, acknowledged this need for harmonization and encouraged representatives of several international organisations – American Association of Pharmaceutical Scientists (AAPS), Applied Pharmaceutical Analysis (APA), Calibration Validation Group (CVG) and EBF – to work with their respective memberships and collaborate on global BMV harmonization efforts. Specifically, both agency representatives said they would be open to h earing from the international scientific community proposals for consistent h armonized regulatory language. The first action in this direction was to pubxad lish the open letter that they had sent to the US FDA and EMA in February 2010, requesting global harmonization of existing or emerging guidances for BMV and sample ana lysis and offering to support the processes needed [2]. In addition, representatives of the above named organizations published two editorials in the same journal discussing the history of BMV and expressing their views on a future harmonization process [3,4]. Concurrent with these publications, the CVG had its fourth workshop on regulated bioanaxad lysis in Montreal. This meeting focussed serixad ous attention on the topic of harmonization of bioanalytical guidelines through a number of speakers from industry and regulatory agencies presenting their ideas and discussing possible future processes with the audience [5]. At the CVG workshop, a unanimous consensus was reached among the five regulatory agencies, the panellists and the international audience that global guidance should be science driven, not prescriptive, and should include rationale behind each requirement to prevent ‘box checking’ by auditors and reviewers of filings. Furthermore, the need to rise above local issues and focus on globally acceptable language will be pivotal to a successful outcome.

Implementing Dried Blood Spot Sampling for Clinical Pharmacokinetic Determinations: Considerations from the IQ Consortium Microsampling Working Group

This paper was developed with the support of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). IQ is a not-for-profit organization of pharmaceutical and biotechnology companies with a mission of advancing science-based and scientifically driven standards and regulations for pharmaceutical and biotechnology products worldwide. Within the IQ, various working groups (WG) have been formed, where the microsampling WG is committed to providing a scientific forum for the advancement of both wet and dry microsampling techniques within the pharmaceutical industry. This first output from the microsampling WG is to summarize and reflect on the current knowledge and opinions on DBS sampling, to stimulate discussion, and to encourage future creative applications of DBS sampling. n nDried blood spot (DBS) sampling has established itself as an innovative sampling technique where wet blood is spotted onto absorbent paper or other paper materials and allowed to dry (1–4). DBS offers several potential benefits inherent to the technique, namely a low blood volume, simplified blood sample collection (5), and convenient sample storage and transfer. In certain applications, DBS sampling has been shown to stabilize certain analytes or metabolites without the addition of chemical modifiers (6–9). DBS has been routinely applied for decades in neonatal screening for phenylketonuria and other congenital metabolic disorders (10). The utility of DBS sampling has also been demonstrated for therapeutic drug monitoring (11) and for epidemiological studies (e.g., HIV and HBV detection/monitoring) (12) due to the practical advantages along with simplified sample collection and handling procedures. Finally, DBS can also be used for quantitative biomarker (PD) assessment from blood, where appropriate. n nHowever, the technique is relatively new to the pharmaceutical industry and to government regulators overseeing new drug applications. Nevertheless, over the past 5 to 7 years, the technique has been extensively evaluated for quantifying drug exposure in nonclinical and/or clinical studies in various stages of drug discovery and development. The ease to collect, transfer, store, and process small volumes of blood samples has generated considerable interest in providing utility in volume-limited situations (e.g., small rodent, human pediatric studies) for toxicokinetic (TK), pharmacokinetic (PK), or pharmacodynamic (PD) sampling. n nDiscovery and nonclinical studies nRodent animal models are typically employed in these studies. The reduced blood volumes required for DBS can enable serial bleeding and, consequently, elimination of satellite animal groups and reduction of compound use. The ability to eliminate the satellite animal groups enables the assessment of exposure and toxic effects within the same animal. Studies involving expensive animal models (i.e., transgenic mice, knock-out mice, humanized mice, etc.) further highlight a persuasive scientific and economic case for DBS sampling since a complete pharmacokinetic profile can be obtained from a single study animal without the need for extra rodents merely for generating exposure data. These are perfectly in line with the principles of the 3Rs: reduction, refinement, and replacement of humane animal research (13–15). With greater emphasis from the regulatory authorities to study new drugs for infants, neonates, and pediatric populations, the requirement to conduct associated nonclinical juvenile rodent toxicity studies serves as an ideal scenario where the advantage of low blood volume in DBS sampling is undeniable. Although the advantages of DBS heavily favor rodent studies, it can also be used to refine non-rodent studies.

Evaluation of dried blood spot (DBS) technology versus plasma analysis for the determination of MK-1775 by HILIC-MS/MS in support of clinical studies

The collection of human blood samples as dried blood spots (DBS) for the pharmacokinetic assessment of investigational drugs in clinical trials offers a number of advantages over conventional plasma sampling, namely, small sample volume, simplified sample handling, and cost-effective shipping and storage. The use of DBS coupled with liquid chromatography–tandem mass spectrometry analysis was evaluated for the quantification of MK-1775, a Wee-1 inhibitor under development as a chemo/radio-sensitizer for the treatment of cancer. The DBS method exhibited an assay performance comparable to that of the existing plasma assay, which is currently used in support of clinical studies. Both assays used the same linear dynamic range of 2–1,000xa0ng/mL, with a lower limit of quantification of 2xa0ng/mL. Based on the intra-day assay validation results, the accuracy of the DBS method ranged from 94.0 to 105.0xa0%, with a coefficient of variation of <4.8xa0%. The blood-to-plasma ratio calculated from the DBS data (blood concentrations) and the plasma data (plasma concentrations) was in good agreement with the one obtained from the in vitro assessment using conventional methodology. No significant hematocrit impact on the assay was observed as hematocrit ranged from 16 to 85xa0%. The correlation between the measured MK-1775 concentrations in plasma and that determined in dried blood spots from oncology patients during the ongoing clinical study was discussed.

Effect of alendronate and vitamin D3 on fractional calcium absorption in a double‐blind, randomized, placebo‐controlled trial in postmenopausal osteoporotic women

Menopause and increasing age are associated with a decrease in calcium absorption that can contribute to the pathogenesis of osteoporosis. We hypothesized that alendronate plus vitamin D3 (ALNu2009+u2009D) would increase fractional calcium absorption (FCA). In this randomized, double‐blind, placebo‐controlled multicenter clinical trial, 56 postmenopausal women with 25‐hydroxyvitamin D [25(OH)D] concentrations of 25u2009ng/mL or less and low bone mineral density (BMD) received 5 weekly doses of placebo or alendronate 70u2009mg plus vitamin D3 2800 IU (ALNu2009+u2009D). Calcium intake was stabilized to approximately 1200u2009mg/d prior to randomization. FCA was determined using a dual‐tracer stable‐calcium isotope method. FCA and 25(OH)D were similar between treatment groups at baseline (0.31u2009±u20090.12u2009ng/mL and 19.8u2009±u20094.7u2009ng/mL, respectively). After 1 month of treatment, subjects randomized to ALNu2009+u2009D experienced a significant least squares (LS) mean [95% confidence interval (CI)] increase in FCA [0.070 (0.042, 0.098)], whereas FCA did not change significantly in the placebo group [−0.016 (−0.044, 0.012)]. After ALNu2009+u2009D treatment, patients had higher 25(OH)D levels (LS mean difference 7.3u2009ng/mL, pu2009<u2009.001). The rise in serum 1,25‐dihydroxyvitamin D3 (pu2009<u2009.02) and parathyroid hormone (pu2009<u2009.001) were greater in the ALNu2009+u2009D group than in placebo‐treated patients. ALNu2009+u2009D was associated with an increase in FCA of 0.07. To our knowledge, there is no other trial showing such a marked rise in calcium absorption owing to treatment with a bisphosphonate or owing to a small rise in 25(OH)D. This unique response of ALNu2009+u2009D is important for the treatment of osteoporosis, but the exact mechanism requires further study.

An Integrated Strategy for Implementation of Dried Blood Spots in Clinical Development Programs

Dried blood spot (DBS) sample collection has gained increased interest across the pharmaceutical industry as a potential alternative to plasma for pharmacokinetic (PK) evaluations. However, regulatory guidelines and examples of late-stage clinical trial applications in the literature are lacking. This paper communicates Merck’s strategy for the implementation of DBS exemplified by experience on a late-stage program (MK-8931). In this program, DBS was proposed as the sole matrix for phase 3 studies to decrease logistical burden in an aging target patient population (Alzheimer’s disease). In vitro and bioanalytical tests demonstrated initial method feasibility and suitability for further evaluations in the clinic. An in vivo dataset was developed initially in healthy subjects (phase 1 study) and then in patients (phase 2/3 study) to establish a quantitative relationship between the blood and plasma concentrations (bridging dataset) using descriptive and population PK analyses. This allowed for PK conclusions to be seamlessly drawn across the clinical program without impact from the choice of matrix. This integrated information package (in vitro, bioanalytical and clinical) was presented to major regulatory agencies (FDA and EMA) for regulatory input. Based on this package, regulatory concurrence was gained on accepting DBS as the sole matrix in late-stage clinical trials.

Bioavailability of Alendronate and Vitamin D3 in an Alendronate/Vitamin D3 Combination Tablet

These studies were designed to demonstrate that the alendronate (ALN) component of an ALN/vitamin D3 combination tablet was bioequivalent to the 70‐mg ALN tablet and that the pharmacokinetic parameters of vitamin D3 were similar with or without ALN. These were open‐label, randomized, 2‐part, 2‐period, crossover studies. In part I, participants received either a single combination tablet or ALN 70 mg. In part II, participants received either a single combination tablet or vitamin D3 alone. Results from part I showed that the geometric mean ratio (GMR) for total urinary excretion of ALN for both studies fell within the prespecified bioequivalence bounds. Results from part II showed that the pharmacokinetic profiles of vitamin D3 with or without ALN were also similar. The combination tablets are bioequivalent to the ALN 70‐mg tablet with respect to ALN bioavailability. The bioavailability of vitamin D3 is similar in the combination tablets and when administered alone. No serious adverse experiences were reported.

Small Molecule Specific Run Acceptance, Specific Assay Operation, and Chromatographic Run Quality Assessment: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Teams

Consensus practices and regulatory guidance for liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays of small molecules are more aligned globally than for any of the other bioanalytical techniques addressed by the Global Bioanalysis Consortium. The three Global Bioanalysis Consortium Harmonization Teams provide recommendations and best practices for areas not yet addressed fully by guidances and consensus for small molecule bioanalysis. Recommendations from all three teams are combined in this report for chromatographic run quality, validation, and sample analysis run acceptance.